The present invention relates generally to a method for quantitating dsDNA in a buffered solution, and more particularly to a method of determining a dilution factor for fluorometric quantitation of dsDNA in solution.
The quantitation of dsDNA in a sample may be required for prescreening purposes during DNA typing analysis, for analysis of microorganisms in environmental samples during forensic investigations, for detecting pathogens in food and food crops, for biological warfare agent detection, and for other important applications. Efficient methods for selectively quantitating double stranded DNA (dsDNA) are required for a thorough analysis of cells and microorganisms from the body, from marine environments such as the ocean and the seas, from plant environments such as soil and sediment, etc.
Various methods are used to extract dsDNA from these environments. One strategy involves separating cells or microorganisms from a sample taken from a particular environment, and then lysing the cells to release dsDNA. Another strategy involves lysing the cells or microorganisms in a sample that includes the environmental matrix they have been taken from, and then separating the dsDNA from the matrix. Lysing procedures can include incubating the sample with a detergent, freeze thawing the sample, homogenizing the sample in bead mill, and other steps that rupture the cell wall or cell membrane to release the enclosed dsdNA. Extraction procedures typically provide only small amounts of dsDNA. However, the extracted dsDNA can be amplified by PCR (polymerase chain reaction), a method that uses extracted strands of dsDNA as templates from which exact dsDNA copies are made. This way, an adequate supply of dsDNA is available for analysis. Since dsDNA extracts are generally contaminated with materials that inhibit PCR, adequate removal of these contaminants is required prior to PCR. Soil or sediment extracts, for example, include co-extracted humic acids that can interfere with PCR even if present in very small concentrations.
In addition to the adequate removal of contaminants, the quality and quantity of dsDNA prior to PCR should be known. Solution extracts generally contain proteins that are coextracted with the dsDNA that can interfere with PCR. Spectrophotometric analysis is a commonly used technique to determine the relative amounts of dsDNA and protein in an aqueous extract. In solution, dsDNA has a maximum absorption at a wavelength of 260 nanometers (nm) while proteins absorb light strongly near about 280 nm. After preparing a solution extract, the absorption intensities at 260 nm and 280 nm are recorded, and the A260/A280 ratio is calculated to provide an estimate of DNA purity. A ratio of about 1.7-2.0 has been reported to indicate xe2x80x9cclean DNAxe2x80x9d. Unfortunately, the absorption measurements themselves that are used to obtain this ratio might not reflect the true concentration or purity of dsDNA in the sample solution because nucleotides, single stranded nucleic acids, and other contaminants can also contribute significantly to the absorption signals.
Fluorometric analysis is another method used to quantitate dsDNA in a sample containing dsDNA. It is a highly sensitive method, and involves adding a non-fluorescent dye to a solution containing dsDNA to produce a highly fluorescent dye-dsDNA complex. The fluorescence intensities for a wide range of concentrations of the complex are measured, and these measurements are used to create a standard curve. The dye is added to a solution containing an unknown amount of dsDNA to form the complex. The fluorescence intensity of this solution is measured, and the standard curve is used for comparison to determine the concentration of the complex in the solution. Some of the dyes used have been described in U.S. Pat. No. 5,436,134 to R. P. Haugland entitled xe2x80x9cCyclic-Subsituted Unsymmetrical Cyanine Dyes,xe2x80x9d and in U.S. Pat. No. 5,863,753 to R. P. Haugland et al. entitled xe2x80x9cChemically Reactive Unsymmetrical Cyanine Dyes and Their Conjugatesxe2x80x9d.
Results of fluorometric analyses must be interpreted carefully since samples prepared for analysis can include contaminants that prevent an accurate quantitation of the dsDNA in the sample. Contaminants such as proteins, for example, can bind to DNA and prevent the dye from forming the dye-dsDNA complex. U.S. Pat. No. 5,824,557 to T. J. Burke entitled xe2x80x9cMethod for Detecting and Quantitating Nucleic Acid Impurities in Biochemical Preparations,xe2x80x9d which issued on Oct. 20, 1998, describes a fluorometric analysis method that uses a detergent to prevent proteins from binding to DNA.
Fluorometric analysis of samples prepared form soil or sediment environment is generally contaminated with humic acids that attenuate the measured fluorescence intensity of the dye/dsDNA complex. xe2x80x9cRapid Method for Fluorometric Quantification of DNA in Soilxe2x80x9d by R. A. Sandaa et al. which was published in Soil Biol. Biochem, 1998, vol. 30, no. 2, pp. 265-268, includes a description of using fluorometric analysis to quantitate dsDNA from soil extracts. A buffered solution containing dsDNA extracted form soil was combined with PicoGreen dye to form the dye-dsDNA complex. The fluorescence intensity of the solution was measured and compared to standards to determine the concentration of dsDNA in the extract. For insufficiently dilute samples, humic acids present in the extract attenuated the fluorescence of the dye-dsDNA complex. To ensure that a sufficiently dilute sample solution was prepared, a dilution series for each soil sample was required.
Although fluorometric analysis an important technique for quantitating dsDNA in the sample solution, and the quantity of dsDNA in a solution is required knowledge prior to PCR amplification, sample solutions often contain contaminants in amounts sufficient to interfere with fluorescence measurements and with subsequent PCR amplification procedures. A method that accurately and efficiently quantifies dsDNA in the presence of contaminants is therefore highly desirable. In order to avoid time lost in transporting samples to a laboratory to be analyzed, it is also desirable that this method be flexible enough for use in the field where the samples are obtained.
Therefore, an object of the present invention is to provide a method for quantitating dsDNA in the presence of contaminants.
Another object of the invention is to provide a method of accurately quantitating dsDAN in a solution that is contaminated with humic acids.
Still another object of the invention is a method for quantitating dsDNA that can be used in the field.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention includes a method for quantitating dsDNA in an aqueous sample solution containing an unknown amount of dsDNA. A first aqueous test solution containing a known amount of a fluorescent dye-dsDNA complex and at least one fluorescence-attenutating contaminant is prepared. The fluorescence intensity of the test solution is measured. The first test solution is diluted by a known amount to provide a second test solution having a known concentration of dsDNA. The fluorescence intensity of the second test solution is measured. Additional diluted test solutions are similarly prepared until a sufficiently dilute test solution having a known amount of dsDNA is prepared that has a fluorescence intensity that is not attenuated upon further dilution. The value of the maximum absorbance of this solution between 200-900 nanometers (nm), referred to herein as the threshold absorbance, is measured. A sample solution having an unknown amount of dsDNA and an absorbance identical to that of the sufficiently dilute test solution at the same chosen wavelength is prepared. Dye is then added to the sample solution to form the fluorescent dye-dsDNA-complex, after which the fluorescence intensity of the sample solution is measured and the quantity of dsDNA in the sample solution is determined.
Once the threshold absorbance of a sample solution obtained from a particular environment has been determined, any similarly prepared sample solution taken from a similar environment and having the same value for the threshold absorbance can be quantified for dsDNA by adding a large excess of dye to the sample solution and measuring its fluorescence intensity.